Our finding that VEGF A induced downregulation
Our finding that VEGF-A induced downregulation of EphB4 is VEGFR2 dependent (Fig. 4A) is not surprising because VEGFR2 is the primary signaling receptor of VEGF-A in EC . However, our finding that VEGF-A induced upregulation of dll4 cb 839 synthesis is not inhibited by VEGFR2 inhibition suggests that a noncanonical VEGF-A signaling pathway that stimulates dll4 may exist. The transient upregulation and then downregulation patterns of neuropilins 1 and 2 induced by VEGF-A stimulation (Fig. 3) may suggest that these receptors may have functions in this pathway, although it is not clear whether they are involved as either arterial or venous markers of phenotype . The VEGF165 isoform binds to neuropilins , and these all function coordinately during angiogenesis and tumor development , , . However, our data do not suggest a clear function for neuropilins in adult EC.
Our data suggest that VEGF induction of EphB4 downregulation and dll4 upregulation are mitogen-activated protein kinase kinase–ERK dependent but PI3K–AKT independent (Fig. 4). Previous studies have uncovered the opposing roles of PI3K and ERK1/2 in artery–vein specification and arteriogenesis. For example, ERK1/2 signaling is critical in both embryonic vascular development  and arterial morphogenesis in adult tissues , , . Deficient ERK1/2 activity results in defective arterial morphogenesis and restoration of which by suppressing PI3K is sufficient for recovery . Conversely, inhibiting PI3K promotes arterial specification, whereas inhibition of ERK inhibits arterial specification, and expression of active Akt promotes venous specification during embryonic development . Our results are consistent with these reports as inhibition of ERK1/2 signaling restores venous identity and inhibits gain of arterial identity; however, inhibition of PI3K–Akt signaling does not appear to be functional in adult EC, which may be an artifact of cell immortalization with middle T antigen . We used immortalized EC because primary mouse venous EC are difficult to keep alive and healthy in cell culture, and immortalized mouse venous EC are useful to understand mechanisms of mouse vein graft adaptation . However, it is possible that examination of primary venous EC may reveal additional pathways that are relevant, such as the PI3K–Akt pathway.
We also show that EphB4 participates in a negative feedback loop to VEGF-A, with reduced EphB4 signaling associated with augmented VEGFR2 expression, downregulation of EphB4, and upregulation of dll4 (Fig. 5). We used EphB4 heterozygous KO EC, isolated from mice heterozygous for EphB4, because homozygous deletion is embryonic lethal . The mutated EphB4 is prevented from membrane insertion , and we have previously confirmed approximately 50% reduction of extracellular EphB4 detection . These cells also show increased VEGF-induced ERK1/2 phosphorylation but not VEGF-induced VEGFR2 phosphorylation, consistent with EphB4 negative regulation of this pathway . However, it is not clear whether this negative regulation is functional in vivo during vein graft adaptation.
Our data suggest that VEGF-A inhibits venous identity in adult EC, recapitulating the change seen during vein graft adaptation in vivo. Vein graft adaptation to the arterial environment may depend on the plasticity of adult EC, and their ability to integrate VEGF signaling pathways, to properly modify the vessel phenotype. In addition, we show that EphB4 is a negative repressor of VEGF-induced reduced EphB4 expression and increased dll4 expression. This finding may provide a basis for EphB4 retention of venous identity in adult EC.
Acknowledgment This work was supported by the National Institute of Health (R01-HL095498 to A.D.), the American Vascular Association William J. von Liebig Award, and with the resources and the use of facilities at the VA Connecticut Healthcare System, West Haven, CT. The authors have no conflicts of interest to declare.